Damage to crops by insects and other pathogens, including viruses, has resulted in substantial economic losses and decreases in annual agricultural production. Man has created and employed a wide range of pesticidal, fungicidal, bacteriocidal and antiviral chemicals in order to reduce the impact of damage from these biological stresses to food and other plant crops. Although some chemicals have been highly effective in reducing insect damage to valuable agricultural crops, many problems remain from the widespread use of many chemicals which limit their practical utility. For example, chemical application typically provides only transient protection and has to be repeated with varying frequencies depending on such factors as the time of application during the growing season, the life-cycle stage of the target organisms, weather conditions, and the skill, knowledge and expertise of the person applying the chemical treatment. Upon deployment, all organisms in the area are typically exposed to chemical protectants, causing damage to beneficial as well as harmful organisms. In addition, many chemicals are toxic to man and animals, and widespread application has had an adverse impact on the environment.
Other attempts to reduce biological stress damage to crops have included selective plant breeding to obtain genetic expression or amplification of natural resistance characteristics. However, desired overall traits may be under the control of many genes and may be difficult to dissociate from undesired traits. A typical undesired result of desired trait expression or amplification by plant breeding techniques is yield depression--an economically undesirable side effect. Accordingly, a strong need exists for new and improved techniques for obtaining expression of natural resistance characteristics in plant crops.
Signals that are released by attacking organisms, such as plant and fungal cell wall fragments, called oligouronides (plant origin) and .beta.-glucans and chitosans (fungal origins), trigger the activation of a spectrum of defense genes that include proteinase inhibitor proteins. It is the combined expression of several of these genes (depending upon the plant) at once that confers resistance against attacking insects, microorganisms and/or viruses. Research on the direct effects of proteinase inhibitors in plant defense against insects has advanced much faster than research on the other defensive chemicals, and the regulation and expression of the proteinase inhibitor genes has provided a model for studying the expression of all of the genes that code for the defensive chemicals.
Proteinase inhibitor proteins are found throughout all life forms and comprise one of the most abundant classes of proteins in the world. The blood of higher animals, for example, contains cumulatively over 200 mg of various proteinase inhibitor proteins per 100 ml serum (Laskowski, M. Jr. and I. Kato, "Protein Inhibitors of Proteinases," Ann. Rev. Biochem. 49: 593-626, 1980). In the plant kingdom most storage organs, such as seeds and tubers, contain from 1-10% of their proteins as inhibitors of various types of proteolytic enzymes (Ryan, C. A., "Proteinase Inhibitors," The Biochemistry of Plants, A Comprehensive Treatise (P. K. Stumpf and E. E. Conn, eds), Vol. 6, pp 351-371, Academic Press, New York, 1981), and some fruits contain up to 50% of their proteins as inhibitors of serine endoproteinases (Pearce, G., D. Liljegren and C. A. Ryan, "Proteinase Inhibitors in Wild Tomato Species," Tomato Biotechnology, (D. J. Nevins and R. A. Jones, eds) pp 139-144, Alan R. Liss, Inc., New York, 1987). Proteinase inhibitors have been identified for all four classes of proteases (serine, cysteine, metallo- and aspartyl-) and, in the case of serine proteinase inhibitors, several non-homologous families have been isolated and characterized (Laskowski, M. Jr., I. Kato, W. J. Kohr, J. S. Park, M. Tashiro and H. E. Whatley, "Positive Darwinian Selection of Protein Inhibitors of Serine Proteinases," Cold Spring Harbor Symposium in Quantitative Biology, in press).
The functions of the inhibitors in nature appear to be twofold: (1) to prevent uncontrolled proteolysis within cells, organelles or fluids where limited proteolysis is important to biochemical or physiological processes, or (2) to protect proteins of cells, fluids or tissues from foreign proteolytic enzymes. The specific roles of most known proteinase inhibitors, however, are not well understood. This reflects a lack of detailed knowledge about how proteolysis is regulated in many, if not most, processes in nature. Mammalian protein digestion and blood clotting are among the most studied and best understood proteolytic systems (Neurath, H., "Evolution of Proteolytic Enzymes," Science 224: 350-363, 1984). The enzymes have been studied for years at the structural and mechanistic levels and more recently at the level of gene regulation. Such extensive detailed information is not available concerning the structure, function and regulation of most other complex proteolytic systems, such as post-translational modification, protein processing, protein turnover, remodeling, etc. (Bond, J. and P. E. Butler, "Intracellular Proteases," Ann. Rev. Biochem. 56: 336-364, 1987). Recently, exciting new insights into intracellular protein turnover in procaryotic and eucaryotic cells have revealed roles for ATP, ubiquitin, and calmodulin and calcium in regulating protein degradation. Relationships are being found between the structures of proteins and their half lives in cells, organelles and fluids (Bond, supra; Rechsteiner, M., S. Rogers and K. Rote, "Protein Structure and Intracellular Stability," TIBS 12: 390-394, 1987; Dice, J. F., "Molecular Determinants of Protein Half-lives in Eukaryotic Cells," FASEB J., 349-357, 1987). The roles, if any, of proteinase inhibitors in most of these processes are poorly understood.
Neurath, supra, has suggested that, in primitive organisms, control of proteolysis was probably accomplished with proteinase inhibitors. Later, as more complex biochemical and physiological systems evolved, so did complex mechanisms to control proteolytic activity, such as zymogen activation and compartmentation, as well as more refined controls by proteinase inhibitors. Plants appear to have retained vestiges of the primitive control mechanisms for proteolysis. Relatively high levels of proteinase inhibitors are synthesized and stored in plant tissues where they can interact with plant pests or pathogens that attempt to consume them (Ryan, supra). The effects of proteinase inhibitors on insect digestive enzymes was first researched by Birk and her associates in the early 1960s (Ryan, supra). From this and other research in many laboratories over the next 20 years, it became clear that the defensive role of proteinase inhibitors was only a part of a complex interaction between the many defensive chemicals that are present or induced in plants and the predators and pathogens that attack the plants. Plants have been at war with their predators for hundreds of millions of years and have evolved various, and sometimes complex, chemical weapons of defense. This arsenal has included inhibitors of the digestive proteolytic enzymes of the attacking pests (Rhoades, D. F. "Evolution of Plant Chemical Defense against Herbivores," Herbivores: Their Interaction with Secondary Plant Metabolites (G. Rosenthal and D. H. Janzen eds) pp 4-55, Academic Press, Inc., New York, 1979).
The digestive processes of higher animals, insects, and microorganisms can vary considerably, both in the classes of the enzymes utilized for protein digestion and in their specificities. Thus, in considering the capability of any proteinase inhibitor in a plant tissue to inhibit a foreign protease, either secreted by a microorganism or released into the digestive tract of a herbivore, the mechanistic class and the peptide bond specificity of the proteinase must be considered as well as structural aspects of the inhibitor that determine its ability to specifically interact with the enzyme (Laskowski, 1980, supra; Laskowski, M. Jr., I. Kato, W. Ardelt, J. Cook, A. Denton, M. W. Empie, W. J. Kohr, J. S. Park, K. Parks, B. L. Schatzley, O. L. Schoenberger, M. Tashiro, G. Bichot, H. E. Whatley, A. Wieczorek and M. Wieczorek, "Ovomucoid Third Domain from 100 Avian Species: Isolation, Sequences, and Hypervariability of Enzyme-Inhibitor Contact Residues," Biochem. 26: 202-221, 1987). The association constant of the interaction must be of sufficient magnitude to effectively inhibit the enzyme. Association constants of most protease-inhibitor interactions are from 10.sup.6 M to 10.sup.10 M, and sometimes higher (Laskowski, 1980, supra).
Inhibitors of serine proteases have received far more interest than the other classes of proteinase inhibitors. Nature has apparently invented serine endopeptidase inhibitors several times, resulting in at least 13 families, as set forth in the following TABLE 1.
TABLE 1 ______________________________________ FAMILIES OF PROTEIN INHIBITORS OF SERINE PROTEINASE ______________________________________ Animals 1. Bovine pancreatic trypsin inhibitor (Kunitz) family. 2. Pancreatic secretory trypsin inhibitor (Kazal) family. 3. Ascaris inhibitor family. 4. Chelonianin family. 5. Serpin family (mechanistically distinct). 6. Hirudin family. Plants 7. Soybean trypsin inhibitor (Kunitz) family. 8. Soybean proteinase inhibitor (Bowman-Birk) family. 9. Potato I family. 10. Potato II family (Inhibitor II family). 11. Barley trypsin inhibitor family. 12. Squash inhibitor family. Microbial 13. Streptomyces subtilisin inhibitor (SSI) family. ______________________________________
None of the families shown in TABLE 1 exhibit homology with any other family. All of the inhibitors of serine proteinases employ the same competitive mechanism of inhibition. Researchers in many laboratories over the past 20 years have contributed to the elucidation of the structure, chemistry, and mechanism of action of the serine proteinase inhibitors (Laskowski, 1980, supra; Laskowski, in press, supra). In brief summary, the side chain of the P.sub.1 residues of the reactive sites of the inhibitors determine their specificities. It is this residue that the enzyme recognizes as a potential substrate. The P.sub.1 residues of the reactive sites of some representative serine proteinase inhibitors are shown in the following TABLE 2.
TABLE 2 ______________________________________ REACTIVE SITES OF PROTEINASE INHIBITORS ENCODED BY VARIOUS INHIBITOR GENES OR cDNAs REACTIVE SITES GENE P.sub.1 --X SPECIFICITY ______________________________________ Inhibitor I Family TOMATO I --Leu--Asp-- CHYMOTRYPSIN POTATO I --Met--Asp-- CHYMOTRYPSIN SUBTILISIN BARLEY C2 --Met--Glu-- SUBTILISIN Inhibitor II Family TOMATO II Domain I --Arg--Glu-- TRYPSIN POTATO II Domain I --Arg--Glu-- TRYPSIN TOMATO II Domain II --Phe--Asn-- CHYMOTRYPSIN POTATO II Domain II --Leu--Asn-- CHYMOTRYPSIN Bowman-Birk Family ALFALFA Domain I --Arg--Ser-- TRYPSIN ALFALFA Domain II --Lys--Ser-- TRYPSIN SOYBEAN Domain I --Lys--Ser-- TRYPSIN SOYBEAN Domain II --Leu--Ser-- CHYMOTRYPSIN ______________________________________
When the side chain of the P.sub.1 residue enters the specificity pocket of the enzyme, about 200 non-covalent Van der Waals and hydrogen bond contacts, involving just a few amino acids, interact at the interface. The cumulative energy of the interactions virtually freezes the two proteins in a stable complex in which the enzyme cannot complete the hydrolysis of the peptide bond, nor can the complex easily dissociate.
The presence of a small percentage of the total dietary proteins as serine proteinase inhibitors can have severe effects on the digestive physiology of animals, including insects. In laboratory animals, trypsin inhibitors can reduce the effective concentration of trypsin that is available to the animal for digestion. This, in turn, lowers the effectiveness of trypsin to activate other proenzymes secreted by the pancreas. Additionally, the trypsin-inhibitor complexes can trigger feedback mechanisms that signal the pancreas to trigger an overproduction of digestive enzymes, while signaling the stomach and brain to reduce the desire of the animal to eat. Prolonged feeding on the inhibitors can lead to the inability to derive amino acids from food as well as the inability to recycle essential amino acids present in the secreted digestive enzymes.
Studies of the effects of dietary proteinase inhibitors on the growth and development of insects, either artificially introduced into defined diets or already in plant tissues, have shown that the native inhibitors can be detrimental to the growth and development of insects from a variety of genera including Heliothis, Spodoptera, Diabiotica and Tribolium (Ryan, supra; Broadway, supra; Rechsteiner, supra). This anti-nutrient property is probably enhanced by, or enhances, other anti-nutrient or toxic chemicals that are part of the array of defensive chemicals of plants. The proteinase inhibitors, while not having an intrinsically high toxicity, therefore provide a set of genes with which to transform plants to study both fundamental and applied aspects of plant defense against herbivores and pathogens.
The large numbers of known, naturally-occurring proteinase inhibitors encompass a wide range of inhibitor specificities. Extensive studies of the relationships of the structures at the reactive sites of avian ovomucoids (serine proteinase inhibitors) from 100 species have revealed that 8 of the 11 amino acids that are involved with the contacts between serine proteinases and their inhibitors are hypervariable (Laskowski, 1987, supra), that is, these amino acids are mutating faster than the rest of the amino acids of the inhibitors. In enzymes, the opposite situation occurs. The active site residues do not mutate as fast as residues that are not involved in the enzyme action. The evidence indicates that some environmental pressures are directing this hypervariability (Laskowski, 1987, supra). One possibility for this phenomenon may be that a natural selection of proteinase inhibitor specificities has taken place in response to a changing spectrum of proteinases of attacking predators and pathogens.
In addition to naturally-occurring proteinase inhibitors, several serine proteinase inhibitor genes and/or cDNAs have recently been isolated and used to transform plants with foreign proteinase inhibitor genes to confer their defensive capabilities. For Example, European patent application Publication No. 0272144 and Hilder, V. A., et al., "A Novel Mechanism of Insect Resistance Engineered into Tobacco," Nature 330:160-163 (1967), disclose a cDNA coding for a cowpea trypsin inhibitor (CpTI) fused with a constitutive CaMV promoter and a nopaline synthase terminator, and the use of the construct to transform tobacco plants. The CpTI trypsin inhibitor was constituitively expressed in the leaves of transformed plants. The transformed plants were said to exhibit a resistance toward Heliothis virescens, the tobacco bud worm, which is an insect pest that ordinarily thrives on tobacco leaves. Sanchez-Serrano, J. J., et al., "Wound-Induced Expression of a Potato Inhibitor II Gene in Transgenic Plants," EMBO J 6:303-306 (1987) discloses transformation of tobacco plants with a wound-inducible potato Inhibitor II gene.
Activation of defensive genes in plants by pathogen and herbivore attacks, or by other mechanical wounding, can result from the action of a variety of signaling molecules that are released in complex temporal patterns following the initial invasion of the tissues (see Darvill, A. G. and P. Albersheim, Ann. Rev. Plant Physiol. 35:243-275, 1984; M. A. Lawton and C. J. Lamb, Mol. Cell. Biol. 7:335-341, 1987; and C. A. Ryan, Ann. Rev. Cell Biol. 3:295-317, 1987). Transport of these signals is mediated locally through intercellular and intracellular fluids that permeate wound or infection sites (Green, T. R. and C. A. Ryan, Science 175:776-777, 1972) or travel systemically through the vascular system of the plants (Kuc, J. and C. Presisig, Mycologia 76:767-784, 1984; M. Kopp, et al., Plant Physiol. 90:208-216, 1990; and K. E. Hammond-Kosack, et al., Physiol. Mol. Plant Path. 35:495-506, 1989). Limited indirect evidence has indicated that signaling may also occur through the atmosphere (Baldwin, I. T. and J. C. Schultz, Science 221:277-279, 1983); D. F. Rhoades in Plant Resistance to Insects, P. Hedin, ed., American Chemical Society, Washington, D.C., 1983; and H. J. Zeringue, Phytochemistry 26:1357-1360, 1987). Whereas several chemicals, including ethylene, have been identified as candidate intracellular signaling molecules for inducible defense genes, no direct biochemical evidence has been presented in the art that would implicate any volatile chemicals besides ethylene as signals that can active plant defensive genes. Ethylene is highly selective in activating defensive genes and only activates chitinase synthesis. Ethylene does not activate the syntheses of other defensive chemicals shown in Table 1 and therefore is not part of the general mechanism for activating defensive genes in plants.
The chemical structure of jasmonic acid is similar to the prostaglandins, important signaling molecules in animals (Samuelson, B. et al., Ann. Rev. Biochem. 47:997-1029, 1978). Jasmonic acid is apparently synthesized from linolenic acid, a fatty acid ubiquitous in plants (Vick, B. A. and D. C. Zimmerman, Plant Physiol. 75:458-461, 1984; and J. M. Anderson in Second Messengers in Plant Growth and Development, Alan R. Liss, Inc., pp. 181-212, 1989). The release of linolenic acid or 3,6,9,12-octadecatetraenoic acid, triggered by the activation of specific lipases in response to pest or pathogen attacks, could lead rapidly to the production of jasmonic acid or methyl jasmonate through the action of enzymes present in the plants. Jasmonic acid may then act as a second messenger molecule in signal transduction pathways leading to defensive gene expression.
Previous studies have shown that methyl jasmonate, or jasmonic acid, when applied directly to plants can produce a variety of responses including growth inhibition (Dalther, W., et al., Planta 153:530-535, 1981); and J. Yamane, et al., Plant and Cell Physiol. 22:689-697, 1981), the promotion of senescence and/or abscission (Ueda, I. et al., Plant Physiol. 66:246-249, 1980; and Curtis, R., Plant Growth Regulators 3:157-168, 1984) as well as the induction of specific leaf proteins in monocots and dicots (Mueller-Uri, J., et al., Planta 176:241-247, 1988; J. M. Anderson, et al, Plant Science 62:45-52, 1989; and P. E. Staswick, The Plant Cell 2:1-6, 1990). There has been no suggestion in the art, however, that jasmonate derivatives could be involved in plant predator defense mechanisms.
In potato and tomato leaves, two small wound-inducible gene families of serine proteinase inhibitors have been identified (Lee, J. S. et al., Proc. Natl. Acad. Sci. USA 83:7277-7281, 1986; and, Cleveland, T. E. et al., Plant Mol. Biol., in press). From wound-inducible mRNAs coding for the two families of inhibitors, cDNAs have been isolated (Graham, J. et al., J. Biol. Chem. 260:6555-6560, 1985; and, Graham, J. et al., J. Biol. Chem. 260:6560-6564, 1985) and utilized as probes to identify the wound-inducible genes in potato and tomato gene libraries (Lee et al., supra. and Cleveland et al., supra). 5' Flanking sequences to the wound-inducible potato inhibitor IIK gene have been operably-linked to give wound-inducible expression of chloramphenicol acetyltransferase (CAT) in transgenic tobacco plants (Thornburg et al., Proc. Natl. Acad. Sci. USA 84:744-748, 1987). However, there has been no suggestion in the art that jasmonate derivatives could be used to induce expression of a foreign gene operably-linked to such a wound-inducible 5' regulatory sequence.